Screening Of High Laccase-producing Mutant Strains By Agrobacterium Tumefaciens-mediated Transformation

Laccase, a copper-containing polyphenol oxidase, which has special catalytic properties in oxidizing aromatic amines and phenolic compounds, is prospective for various applications in different fields such as pulp bleaching in paper, food industry, biological synthesis and bioremediation. Pleurotus ostreatus is a white-rot basidiomycete secreting different enzymes involved in lignin degradation, and also is one of a popular laccase-producing strain under investigated. With the development of molecular biology, using genetic engineering technology to modify the target gene becomes an important method to construct strains in order to meet the demand in industries and agriculture. In this paper, we used Agrobacterium tumefaciens-mediated transformation to construct high yield laccase-producing mutant strains which may provide basic reference in industrial application.The results about the lacccae synthesis ability in different strains showed that laccase activities were almost the same in PDA medium. While laccase activities varied when two inducers were added to the medium. Laccase activity in Pleurotus ostreatus No.5 was greatly induced by copper, the highest activity 329.78 U/mL was obtained on the 6th day of cultivation. Laccase activity in Pleurotus ostreatus No.4 and No.5 were both induced by yeast extract. The highest laccase activities were 613.98 U/mL and 783.61 U/mL respectively. Considered other factors such as growth rate, Pleurotus ostreatus No.5 was chosen to be the tested strain.Classical Agrobacterium tumefaciens-mediated transformation of mushroom is cocultivated of Agrobacterium and fruiting body gill tissue. While the limited factor is a long-time-cultivation and management. Thus we devised an easy and efficient method for Agrobacterium-mediated transformation of the mycelia of Pleurotus ostreatus--nested plates. The advantages of this method are simple operation, short term, and easy to expand the scale of experiment. The result of optimization of co-culture conditions were as follows:the Agrobacterium tumefaciens concentration for OD600= 0.6-0.7, 200 ?mol/L AS in the induction medium, co-culture 3-5 d in 25?.Lots of transformants were got by the method described previously. The first-round screening were used the color reaction between laccase and ABTS. The second-round screening were in shake flask.4 potential high laccase-producing strain were obtained. The laccase synthesis abilities were tested in liquid medium with different inducers. The results showed the laccase activities were increased in different level in PDA medium compare with the strain of wild type. The mutant No.4 and No.6 increased almost 2-fold. When copper was added to the medium to the final concentration of 1 mmol/L, the mutant No.7 showed the greatest increasing in laccase activity, the highest activity 1004.17 U/mL was obtained on the 16th day of cultivation, increasing 13-fold compare to the original level. Mutant No.2 and No.6 showed the highest activities in the present of yeast extract, the highest activities 1022.22 U/mL and 1062.5 U/mL were obtained, increased 13 and 10-fold separately compare to the original level.